While genomic and phenotypic analysis has reached a massive scale with the widespread use of DNA chips and arrays, the world of proteomics remains hindered by the complexity of proteins and the difficulty of their analysis. We seek to create an affordable and easily implementable proteomic tool in the form of low cost "enzyme assay chips" that allow evaluation of enzyme activities at the level of 400 reactions per cm2 or up to 4,000 reactions per slide. Each reaction will be tailored for detection of a specific enzyme activity in the presence of individualized activators, cofactors, and inhibitors of the target enzyme of assay at each sensing element. A novel microfluidics technology has been patented and tested for this application. Specific Aim 1 (design driven) will define the operational/quality control conditions for rapid sensing of enzyme activities on high-density chips using less than 100,000 cells. This aim will also utilize purified enzymes to test and calibrate the enzyme activity-sensing surface. Specific Aim 2 (hypothesis driven) will seek to create an "apoptosis" chip to map apoptotic pathways in cells subjected to several chemical agents. We hypothesize that sub-critical levels of several apoptotic inducers will interact to cause a marked change in the readout of the chip. Specific Aim 3 (design/hypothesis driven) seeks to create de-convolution software to predict hidden variables (concentrations of numerous enzymes) delivered to the sensor surface. We hypothesize that complex patterns of enzyme sensing can be de-convoluted for over 10 enzyme activities in a sample for every 100 enzyme determinations even in the presence of enzyme cross-talk. Phase I activities will lead toward establishing chip chemistries for a commercialized chip to aid in apoptosis, cancer, drug toxicity research, as well as, secondary drug testing and industrial bioreactor monitoring. This will enable Phase II activities that will focus on the development of an automated, yet affordable bench top technology for the utilization. PROPOSED COMMERCIAL APPLICATION: An enzyme assay chip technology will be validated for the assay of numerous intracellular proteases using extremely small cellular or biological samples placed on a sensing chip. This technology can have research, medical, and industrial uses with respoect to the monitoring of intracellular enzyme states and identifying potential drug targets.